In vivo test of Litopenaeus vannamei infected by Vibrio using Moringa oleifera leaf extract

This study aimed to determine the optimal concentration of Moringa oleifera leaf extract as an antibacterial agent on Litopenaeus vannamei larvae that are infected by Vibrio sp. It was conducted in 15 March - 15 April 2019 at PT. Global Gain Superior Seeds, Pantai Cermin, North Sumatera. The research used a completely randomized design (CRD) method with five treatments and four replications. The shrimps infected by Vibrio sp. were immersed into M. oleifera leaf extract in various concentration of (A) 0 ppm, (B) 400 ppm, (C) 600 ppm, (D) 800 ppm, and (E) 1000 ppm for ±15 minutes. The size of shrimp used was post-larvae 8 (length of 3.5-5 mm/ind). The best performance results of vannamei shrimps were obtained in treatment E (1000 ppm), namely length growth of 16.75 ± 1.16 cm, weight growth of 0.92 ± 0.89 g and survival rate of 91±7.87%.


Introduction
Vannamei shrimp (Litopenaeus vannamei) is an aquaculture commodity with high economic value. However, bacterial disease attacks are often observed as an obstacle in shrimp cultivation, causing big losses in both the aquaculture area and in hatcheries. Bacterial diseases caused by Vibrio sp. are the most serious and often lead to mass mortality on the larvae of vannamei shrimps. According to Sharma et al. (2010), farmers claim that the shrimp disease most frequently reported is vibriosis caused by Vibrio sp. infection. The research of Liu et al. (2019) also reported that the Vibrio bacteria group is the disease causing pathogens for shrimps.
Generally, Vibrio is a pathogenic bacteria species that cause mass mortality of panaeid shrimps in hatcheries and pond aquaculture (Amatul-Samahah et al. 2020). In addition, infection by Vibrio bacteria sometimes becomes primary infection that would then stimulate other pathogens to infect shrimp. Liu and Chen (2004); Octaviana et al. (2014) mentioned that shrimp production can be hampered by Vibrio spp. where the disease infection caused by these bacteria frequently results in secondary infection during the cultivation activities. The devastating impact of simultaneous infection by multiple pathogens can increase shrimp mortality.
Antibiotic is usually used by shrimp farmers to handle the infectious microorganisms on the site. However, the use of antibiotics would result in negative impacts, such as residue accumulation in the shrimp meat that could be harmful for consumers. It is also mentioned by Maryani et al., (2002) that antibiotics given through feed, immersion, or injection would leave residues that can accumulate in the body of fishes. The frequent use of antibiotics to cope with disease caused by bacteria in shrimp aquaculture is also becoming less effective, since it generated increased resistant among the pathogens, and also leads to environment pollution (Sukenda et al., 2015). Therefore, the frequent use of antibiotics is not justified in aquaculture activities. As a result, interest is increasing on the use of natural ingredients/herbs as antibacterial agents with less negative effects both Depik Jurnal Ilmu-Ilmu Perairan, Pesisir dan Perikanan Volume 10, Number 2, Page 86-90 Arisa et al. (2021) on the shrimp themselves, and on the people who consume them. Purnamasari et al. (2010) reported that Indonesian people have long considered that natural ingredients/herbs could be used to treat various shrimp diseases and rarely cause adverse side effects compared to the use of chemicals. One of the herbal ingredients that can be used as medicine is moringa leaves (Moringa oleifera). According to Rani et al., (2018) M. oleifera contains a variety of phytoconstituents such as alkaloids, saponins, tannins, steroids, phenolic acids, glucosinolates, flavonoids and terpenes. The diversity of phytochemicals in this genus can contribute to a variety of pharmacology uses. Furthermore, Aminah et al., (2015) stated that the bioactive compounds in M. oleifera has pharmacological properties and M. oleifera leaves have high antioxidant and antimicrobial content. Suhartono et al. (2019) reported that the ethanolic extract of M. oleifera leaves contains flavonoids, tannins, terpenoids, steroids, and alkaloids. Pratama et al. (2018) reported that using the extract of M. oleifera as immunostimulants can enhance the immune response of vannamei shrimp with the best conditions in the extract treatment of 40 mg/L. Based on the reports by researchers about the content of the leaves of M. oleifera, there is research potential on the effect of M. oleifera extract through in vivo test on phatogen infected L. vannamei. Therefore, this study aims to determine the optimal concentration of M. oleifera leaf extract to act as antibacterial agent on the shrimp larvae of L. vannamei that has been infected Vibrio sp. bacteria.

Materials and Methods Location and time of research
The research was conducted in March 15-April 15 2019 at PT. Global Gene Superior Seeds, Pantai Cermin, North Sumatra. The tools used were autoclave, magnetic stirrer, loop needle, 12 L jar, blender, hot plate, pH meter, thermometer, DO meter, scale, refractometer. While the materials used in this study were Moringa oleifera leaf extract, Vibrio sp. Bacteria, TCBS agar medium, vaname shrimp larvae (Litopenaeus vannamei), ethanol, feed and filter paper.
The experimental design used a completely randomized design (CRD) with 5 treatment levels and 4 replications, namely treatment A (concentration of 0 ppm M. oleifera extract), treatment B (concentration of 400 ppm of M. oleifera extract), treatment C (concentration of 600 ppm of M. oleifera extract), treatment D (concentration 800 ppm M. oleifera extract), and treatment E (concentration 1000 ppm M. oleifera extract).

Test shrimp
The test shrimps used were 8 days old (PL 8) size 3-5 mm, with a total of 2,000 individuals and each container was stocked with 100 shrimp. The test shrimps were taken from the same tub and were infected with the Vibrio sp.. The test shrimp were then soaked in their respective treatments of M. oleifera leaf extract for ± 15 minutes, after which they were then kept for 14 days. The calculation of Total Vibrio Count (TVC) on the test shrimp was carried out after soaking with M. oleifera leaf extract.

Extracting Moringa oleifera leaf
The leaves of M. oleifera were dried at room temperature for about 4 days under supervision. The dried samples (simplicia) were cut into pieces and then mashed using a blender to become simplicia powder. The simplicia powder was weighed at 130 g and put into an Erlenmeyer flask. Then immersion (maceration) was done with 2 liters of ethanol solution and soaked for 24 hours. After 24 hours, the solution was filtered using a filter and evaporated using an evaporator (Farika et al., 2014;Kenconojati dan Rukmana, 2019).

Making TCBS media
TCBS powder was put in an Erlenmeyer flask (as much as 14,08 g), and then dissolved by adding 160 mL of distilled water and then heated until boiling on the hot plate while homogenized using a magnetic stirrer. Then, the media was sterilized using an autoclave at 121 0 C with a pressure of 15 1bs for 15 minutes. TCBS media was then used to grow the Vibrio sp. bacteria.

Data analysis
Research data from the in vivo test of Litopenaeus vannamei infected with Vibrio using Moringa oleifera leaf extract were analyzed by ANOVA using the SPSS software program and if the effect was significant, it was followed by a Duncan test. Data were calculated based on the formula in Effendie (1979), which includes weight growth (GR), length growth (P) and survival (SR). In addition, the total bacterial colony (Total Vibrio Count) was also calculated using the formula from Wiyatanto et al. (2020). Survival rate SR = (Nt-N0) x 100% Where: SR= Survival rate (%), Nt = Number of shrimp death during study (ind), N0= Number of shrimp at the start of study (ind).

Total vibrio count (TVC)
Where: ∑c = Number of colonies on all plates count; n1 = The number of plates at the first dilution count; n2 = The number of plates at the second dilution count d = The first dilution calculated.

Results
The result of the research on the growth of vannamei shrimp larvae treated with different concentrations of Moringa leaf extract (M. oleifera) were tested by ANOVA and showed no effect (P>0.05). while the survival test and the total vibrio count of vannamei shrimp larvae showed an effect (P<0.05). The research data can be seen in Table 1.

Discussion
The use of antimicrobial therapy is considered to prevent existing microbial diseases, usually used for healing, where testing is carried out on infected animals in the laboratory (Ibrahim et al., 2020). Result of in vivo test of vannamei shrimp (Litopenaeus vannamei) soaked in Moringa oleifera leaf extract can be seen in Table 1. Maslang et al. (2018) research states that feed substitution of M. oleifera powder in feeds on the survival growth and feed conversion for tilapia resulted in an increase of survival rate and decreased feed conversion.
The results of the immersion study of M. oleifera extract showed an effect on the survival of vannamei shrimp larvae (P<0.05), where the highest shrimp survival rate was 91% in treatment E (concentration of 1000 ppm M. oleifera extract). The increase in survival rate of shrimp larvae is suspected to have been cause by the antibacterial compounds contained within the M. oleifera extract that plays a role in inhibiting the growth of Vibrio sp. pathogen. According to Savitri et al. (2018) M. oleifera leaves contain antibacterial compounds such as suckedonins, triterpenoids and tannins which have action mechanisms that damages bacterial cell membranes. Research by Dima et al. (2016) said that M. oleifera leaves have compounds that can inhibit bacterial growth and the concentration differences in Moringa leaf extract affect the inhibition of growth of Escherichia coli and Stapylococcus aureus bacteria, where the higher the concentration given, the greater the antibacterial activity that inhibit bacterial growth.
The growth and development of vibrio bacteria did not properly occur in treatment E. It was observed that the number of Total Vibrio Count (TVC) in this treatment reached the least growth compared to the other treatments, namely 410 x 10 6 Cfu mL -1 (Table 1). Low TVC in this treatment shows that the immersion of the vannamei shrimp larvae infected with the Vibrio sp. with high concentrations of M. oleifera leaf extract have resulted in the inhibition of bacterial growth. Futhermore Valdez-Solana et al. (2015) said that the leaves of M. oleifera contain falvonoids and phenolic acids which act as antioxidants.
In addition, M. oleifera leaf extract is known to inhibit the formation of vibrio bacteria biofilm. Research by Suhartono et al. (2019) reported that M. oleifera leaf extract significantly inhibited V. alginolyticus biofilms formation in vitro. Biofilms are nutrient traps for the growth of microorganism populations and help prevent the release of cells from the surface of living or inanimate objects. The surface where the biofilm attached is an important habitat for microorganisms because nutrients can be trapped on this surface. Microorganism or biofilms in a system can be destroyed chemically. Other studies regarding the use of M. Oleifera leaves that have been carried out include Farika et al. (2014) who stated that M. oleifera leaf extract can be used as a control for Argulus sp. in Carrasius auratus auratus with the best treatment at the highest concentration 62,5 mgL -1 . Shailemo et al. (2016), reported that M. oleifera extract has antibacterial activity against Bacillus cereus, Enterococcus faecalis and Escherichia coli, with an inhibition zone between 7 -9 mm, at a concentration of 50 mg mL -1 . Futhermore Widowati et al. (2014) also tested the antibacterial activity of M. oleifera leaves extract against spoilage bacteria of fresh fish (Pseudomonas aeruginosa) where the best tratment was shown to be at the highest concentration of 100%.
Depik Jurnal Ilmu-Ilmu Perairan, Pesisir dan Perikanan Volume 10, Number 2, Page 86-90 Arisa et al. (2021) Clinical symptoms of vannamei shrimp larvae stricken with vibriosis disease at the time of study were body weakness, dull appearance of shrimps, inactive movement and decreased appetite. According to research by Sarjito et al. (2015) clinical symptoms of shrimps affected by vibriosis are the reddening of the body (carapace), melanosis of the skin, necrosis of the tail, flushed swimming and walking legs and a reddened hepatopancreas that tends to darken. Vibrio bacteria in aquaculture media are normal flora and they generally thrive when there is an imbalance. Arias-Moscoso et al. (2018) said that when the balance between bacterial communities is disturbed, vibrio bacteria become opportunistic pathogens.
Water quality parameters during shrimp rearing (temperature, pH, DO and salinity are still within the tolerance of optimal shrimp growth. The results of observations in all treatments showed that the temperature ranged between 26-28 o C, pH 8.0-8.3, DO 6-6,6 mgL -1 and salinity 29-30 ppt. Research by Fuady et al. (2013) stated that the management of water quality on survival rates and growth rates of vannamei shrimp needs the range of temperature 26-29 o C, pH 6-8, DO 5-6 mgL -1 and salinity 30 ppt.

Conclusion
Soaking Moringa oleifera leaf extract in vannamei shrimp larvae (Litopenaeus vannamei) infected with Vibrio harveyi at a concentration of 1000 ppm can inhibit bacterial growth and increase the survival of shrimp larvae